Disodium L-cystinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-13 to 2012-01-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Adopted 22 July 2010

Deviations:

yes

Remarks:

The relative humidity in the animal room was between approx 45 - 78 % instead of 45 - 65 % for several hours

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cystine

EC Number:

200-296-3

EC Name:

Cystine

Cas Number:

56-89-3

Molecular formula:

C6H12N2O4S2

IUPAC Name:

cystine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity test date: > 98.5 %

RADIOLABELLING INFORMATION
- Substance: ³H-Methyl thymidine (Hartman Analytic MT6032, aqueous solution)
- Radiochemical purity: not specified
- Specific activity: 74 GBq/mmol (2 Ci/mmol), 37 MBq/mL (1 mCi/mL)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not reported
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated by the sponsor
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was placed into a mortar on a tared balance and propylene glycol was added to achieve the required test item concentration. The different test item concentrations were prepared individually and made freshly before each dosing occasion. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
- Preliminary purification step (if any): not reported
- Final dilution of a dissolved solid, stock liquid or gel: 5, 10, and 25% (w/w) in propylene glycol

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

OlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 10-11 weeks (pre-test); 8 weeks (main study)
- Weight at study initiation: within the range commonly recorded for animals of this strain and age
- Housing: all animals belonging to the same experimental group were kept in one cage: Makrolon Type II / III, with wire mesh top (EHRET GmbH)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories B.V.)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing under test conditions after health examination.
- Indication of any skin lesions: Not specified; only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 45-78 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): artificial light 6 am to 6 pm

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5, 10, 25% (w/w) test item in vehicle

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25 % (w/w) suspension in propylene glycol
- Irritation: To determine the highest non-irritant test concentration that did at the same time not induce signs of systemic toxicity, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25%, once daily, each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible reddening of the ear skin. Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. At the tested concentrations the animals did not show any signs of local skin irritation.
- Ear thickness measurements: Prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch and were immediately pooled per animal and weighed using an analytical balance.
- Erythema scores: No visible erythema on either tested animal at any day of observation.
- Systemic toxicity: At the tested concentrations the animals did not show any signs of systemic toxicity.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
1) Exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index
2) Data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression
- In additon to the sensitising reactions observations of mortality/viability, body weight, ear thickness, ear weights, and clinical signs (local and systemic) were recorded during the test and observation period

TREATMENT PREPARATION AND ADMINISTRATION:
- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations of 5, 10, and 25% (w/w) in propylene glycol. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
- Administration of ³H-Methyl Thymidine: Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.1 µCi of ³HTdR (equivalent to ³HTdR 80.5 µCi/mL) were injected into each test and control mouse via the tail vein.
- Determination of incorporated ³HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH) and thoroughly mixed. The level of 3HTdR incorporation was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).

Results and discussion

Positive control results:

valid

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Variability:

SD: 272.0

Test group / Remarks:

Vehicle control

Results 2

Key result

Parameter:

SI

Value:

0.98

Variability:

SD: 251.3

Test group / Remarks:

5% L-Cystine

Results 3

Key result

Parameter:

SI

Value:

1.24

Variability:

SD: 188.1

Test group / Remarks:

10% L-Cystine

Results 4

Key result

Parameter:

SI

Value:

1.02

Variability:

SD: 250.4

Test group / Remarks:

25% L-Cystine

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : The proliferative response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

DETAILS ON STIMULATION INDEX CALCULATION : The ratio of ³HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals

EC3 CALCULATION : The EC3 value could not be calculated, since all S.I.'s are below the threshold value of 3.

CLINICAL OBSERVATIONS: No symptoms of local toxicity at the ears of teh animals and no systemic findings were observed during study period.

BODY WEIGHTS: Body weight of the animals recorded prior to first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

No death occurred during the study period

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on OECD guideline test 429 Local Lymph Node Assay with mice, the test item L-Cystine was not a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of L-Cystine following OECD guideline 429, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/w) in propylene glycol by topical application to the dorsum of each ear for three consecutive days. A control group was treated with the vehicle only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Single cell suspension of lymph node cells were prepared from pooled auricular lymph nodes and the proliferative capacity of the lymph node cells was determined.

In this study Stimulation Indices of 0.98, 1.24, 1.02 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in propylene glycol, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The test item, L-Cystine, was not a skin sensitiser under the test conditions of this study.